Glutathione S.transferases (GST) are important in the detoxification of xenobiotics, catalyzing the nucleophilic attack by the thiol group of glutathione on the xenobiotic substrate. Since they catalyze the inactivation of several known carcinogens, these enzymes can provide a defense against carcinogenesis. On the other hand, the elevation of GST levels in solid tumors appears to be a major factor in the development of resistance to treatment with cytotoxic agents. The GSTs are grouped into at least five different gene families based on sequence similarity and substrate specificity; e.g., the 1-1 isozyme confers the greatest cellular resistance to the anti-cancer drugs, chlorambucil and melphalan, while the 3-3 isozyme confers the most increase in resistance to cisplatin. The complete amino acid sequences have been determined for the major GSTs of mammalian liver, and three-dimensional structures have recently been reported for crystals of the 3-3 isozyme of the mu class, of two pi class enzymes and of the 1-1 isozyme of the alpha class. However, important questions remain about which amino acid residues contribute to the binding of the xenobiotic substrate, how these residues determine the substrate specificities of the various isozymes of GST, and whether a given enzyme has more than one type of xenobiotic substrate site. We will examine isozyme 1-1 of rat liver as representative of the alpha family, and isozyme 3-3 of rat liver as an example of the mu family of GSTs. These isozymes differ in substrate specificity and comparison of their sequences reveals 87% identical plus similar residues within the mu family but only about 30% between the alpha and mu families. Our studies of the active sites of these enzymes while in solution will be complementary to and will be compared by computer modeling to structures of the protein crystals using the X-ray coordinates. We propose initially to use affinity labeling to effect specific chemical modification and identification of amino acid residues in the region of the catalytic sites of these isozymes. A series of novel reagents will be synthesized based either on the structure of selected xenobiotic substrates or on that of glutathione and featuring reactive electrophilic or photoreactive functional groups capable of covalently labeling amino acid side chains once the reagent binds at the active site. Radioactive precursors are available to synthesize labeled reagents to facilitate isolation of the modified amino acids. Once the modified amino acids have been identified, we will use site-directed mutagenesis to construct appropriate mutant 1-1 and 3-3 enzymes, which will be expressed and characterized to test the function of the target amino acids. This study aims to provide the knowledge base for rational design of inhibitors specific for particular xenobiotic substrate sites for GST for use in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs.